Coagulation factor IX (FIX) is a vitamin K-dependent coagulation factor with structural similarities to factor VII, prothrombin, factor X, and protein C. The circulating human FIX zymogen consists of 415 amino acids divided into four distinct domains comprising an N-terminal gamma-carboxyglutamic acid rich (Gla) domain, two EGF domains, and a C-terminal trypsin-like serine protease domain. Activation of FIX occurs by limited proteolysis at Arg145-Ala146 and Arg180-Val181 releasing a 35 amino acid activation peptide (Schmidt A E, and Bajaj S P (2003) Trends in Cardiovascular Medicine 13, 39-45). Wild-type human coagulation factor IX (SEQ ID NO: 1) has two N-glycosylation sites (N157 and N167).
The half-lives of some proteins can be prolonged by adding N-glycans at amino acid positions that are not glycosylated in the wild-type protein (reviewed by Sinclair A M, and Elliott, S (2005) Journal of Pharmaceutical Sciences 94, 1626-1635). N-glycans are attached to proteins by eukaryotic cells producing the protein. The cellular N-glycosylation machinery recognizes and glycosylates N-glycosylation signals (N-X-S/T motifs) in the amino acid chain, as the nascent protein is translocated from the ribosome to the endoplasmic reticulum (Kiely et al. (1976) Journal of Biological Chemistry 251, 5490-5495; Glabe et al. (1980) Journal of Biological Chemistry 255, 9236-9242). Thus, glycoengineered proteins can be produced by introducing mutations that add N-glycosylation sites to the amino acid sequence of the protein. This principle has been employed to obtain longer-acting second generation erythropoietin (Aranesp®, Amgen). This kind of glycoengineering is very attractive in terms of production of the biopharmaceutical, since the final prolonged protein is secreted to the medium of the producer cells. Thus, unlike PEGylation, glycoengineering does not complicate and increase the cost of downstream processing. Furthermore, hyperglycosylation may shield protein epitopes (Cheng-Mayer et al. (1999) Journal of Virology 73, 5294-5300) and reduce aggregation by increasing the solubility of the protein (Song et al. (2001) FEBS Letters 491, 63-66). In effect, glycoengineering may also improve recombinant proteins by decreasing their immunogenicity, thus reducing the risk that patients develop neutralizing antibodies against the protein.
Interestingly, the influence of N-glycans on clearance varies among different proteins. Several proteins are not influenced by removal or addition of N-glycans. In contrast, some proteins are cleared faster in the absence of their N-glycans and as mentioned above, the clearance of some proteins can be delayed by addition of extra N-glycans (Elliott et al. (2003) Nature Biotechnology 21, 414-421; Perlman et al. (2003) Journal of Clinical Endocrinology and Metabolism 88, 3227-3235). The mechanisms by which N-glycans influence the clearance of some proteins are unknown and may vary between the proteins. For follicle stimulating hormone, reduced renal clearance due to increased size and increased negative charge from sialic acids has been proposed to explain the delay in clearance induced by extra N-glycans (Perlman et al. (2003), supra). The proteins that are known to be prolonged by addition of extra N-glycans are mostly relatively small proteins, which is in agreement with an effect on renal clearance. For erythropoietin, however, solid evidence for an important role of either renal or hepatic clearance remains to be presented. Intracellular degradation of erythropoietin internalized by cells in the bone marrow after binding to the erythropoietin receptor has been suggested as the major mechanism for clearance of circulating erythropoietin (reviewed by Jelkman, (2002) European Journal of Haematology 69, 265-274). The affinity of longer-acting hyperglycosylated erythropoietin to the erythropoietin receptor is reduced compared to wild-type erythropoietin (Elliott et al. (2004) Experimental Hematology 32, 1146-1155), and recent evidence suggests a link between the reduced receptor affinity and slower receptor mediated degradation, leading to a longer circulatory half-life (Gross and Lodish, (2006) Journal of Biological Chemistry 281, 2024-2032). Interestingly, the reduced receptor binding by hyperglycosylated erythropoietin appears to result from the increased sialic acid content (Elliott et al. (2004) Experimental Hematology 32, 1146-1155). The in vitro activity of hyperglycosylated erythropoietin is significantly reduced compared to wild-type erythropoietin, and it is known to a person skilled in the art that a considerable reduction in specific activity must be expected when N-glycans are introduced in proteins at sites that are not glycosylated in the wild-type protein.
US 2003/0036181 (Maxygen, Inc.) describes the addition of glycosylation sites to polypeptides. EP 0640619 (Amgen Inc.) describes erythropoietin analogs with additional glycosylation sites. Mimuro et al. (2004) Journal of Thrombosis and Haemostasis, 2, 275-280 describes human coagulation factor IX with the mutation A262T introducing a N-glycosylation site at amino acid position 260.
There is thus a great need for providing an improved variant of human factor IX which demonstrates an increased in vivo circulatory half-life but without dramatically reducing the proteolytic activity or clot activity when compared with wild-type human factor IX.